WO2003087293A1 - Appareil et techniques d'analyse biochimique - Google Patents

Appareil et techniques d'analyse biochimique Download PDF

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Publication number
WO2003087293A1
WO2003087293A1 PCT/GB2003/001453 GB0301453W WO03087293A1 WO 2003087293 A1 WO2003087293 A1 WO 2003087293A1 GB 0301453 W GB0301453 W GB 0301453W WO 03087293 A1 WO03087293 A1 WO 03087293A1
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WIPO (PCT)
Prior art keywords
electrode
well
sample
mediator
biological sample
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PCT/GB2003/001453
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English (en)
Inventor
Nicholas Paul Cassells
Original Assignee
Lgc Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lgc Limited filed Critical Lgc Limited
Priority to AU2003222593A priority Critical patent/AU2003222593A1/en
Publication of WO2003087293A1 publication Critical patent/WO2003087293A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48735—Investigating suspensions of cells, e.g. measuring microbe concentration

Definitions

  • the present invention relates to apparatus, methods and means for biochemical analysis, more particularly for analysis which monitors the response of one or more biological samples to one or more test agents.
  • the invention may be useful for e.g. studying the effects of prospective pharmaceuticals or other chemicals on biological systems, assaying environmental samples for the presence of pollutants, and clinical analysis of biological specimens for e.g. toxins and pathogens.
  • EP 0 242 225 A2 describes a prior art system for detecting pollution in a continuous stream of aqueous liquid.
  • An electron transfer mediator is added to the liquid, and the resulting mixture is passed to a sensor chamber containing bacteria.
  • An activity of the bacteria is stimulated, and the level of that activity is measured at an electrode by means of electron transfer from the bacteria, to the electrode, by the mediator.
  • any device which monitors, in parallel, the response of a plurality of biological samples to a particular test agent.
  • a more general limitation of the prior art is in respect of the provision of apparatus, methods and means for monitoring biological cells, or components thereof, in states of organisation that are conducive to their proper functioning. For example, it may be necessary for the proper metabolic functioning of certain types of cell for the cells to be grown on a particular substrate. Furthermore, the mode of responsiveness of a population of a first variety of developmentally ' differentiated cells to an external stimulus, e.g. to a pollutant, may be conditional on the positioning of the cells in proximity to a population of a second variety of developmentally-differentiated cells.
  • This situation is a simple manifestation of, or an analogy to, the more complex interdependency of cell varieties in a biological tissue, and/or the further interdependencies introduced by the arrangement of several biological tissues within a biological organ. Consequently, depending on the purpose for which the interaction of a biological sample with a test agent is to be monitored, and the particular biological sample and test agent concerned, it may be necessary to control the spatial arrangement and relative positioning of one or more populations of cells within a biological sample, with respect to one or more components of the test apparatus and, in the case of multiple populations of cells within the sample, with respect to one another. It may be necessary to study samples of a complete tissue or a complete organ. In relation to electrochemical methods of monitoring the function of cells, tissues and organs, e.g.
  • the biological sample may be positioned sufficiently far away from the electrode that all the components of the sample are held at a similar distance from the electrode, and effectively interact equivalently with the electrode via the electron transfer mediator. This may improve the consistency of the results obtained, and the correlation of those results with data that have been obtained from in vivo assays.
  • apparatus and methods may be devised in which the biological sample may be removed for a time, once or repeatedly, from the monitoring environment. This may be beneficial for the reasons that are described elsewhere herein.
  • the region around the electrode need not come into direct physical contact with the biological sample, which may enable the electrode and its environment to be regenerated effectively for subsequent reuse.
  • the performance of the monitoring system as a whole may be optimized by varying the relative positioning of, optionally including the distance between, the biological sample and the electrode: operational parameters including the fluid dynamics and the rate of electron transfer by the mediator may be adjusted in this way.
  • the present invention provides an apparatus for monitoring how a plurality of biological samples responds to one or more test agents, the apparatus comprising a plate having a plurality of wells, each of which is associated with a working electrode which is exposed to its interior.
  • the plurality of wells may be arranged in the plate in a regular pattern, e.g. as a series or array. It may comprise any number of wells, each of which may be of any convenient size and shape.
  • the apparatus is adapted for use in electrochemistry, e.g. for use in cyclic voltammetry, potentiometry and/or amperometry, e.g. for use in chemically mediated amperometry.
  • a chemical mediator typically a redox coupling agent in its oxidised form
  • the mediator then transfers the electrons to a working electrode poised at a sufficient oxidising potential with respect to a reference electrode, and the resulting "current" is monitored.
  • the chemical mediator now re- oxidised, returns to the sample and the process is repeated.
  • the apparatus of the invention provides for an assay system for the simultaneous analysis of a plurality of biological samples.
  • the system therefore has a high throughput, relative to the disclosures of the prior art. It is also very versatile.
  • a user can investigate, in parallel, the responses of a range of biological samples (e.g. a range of different bacterial, plant and/or animal cells) to any given test agent.
  • the different biological samples may be chosen so as to be relevant to the investigation in question.
  • a given test chemical is suspected of being toxic to humans, but the target organ is unknown
  • a range of human cells from e.g. the liver, kidney, gut, lung, muscle, skin, blood etc. can be tested simultaneously, in order to determine which organ is (most) affected.
  • an environmentally relevant chemical may be tested against a range of micro-organisms, in order to determine which (if any) species is at most risk, should a release of the chemical into the environment occur (or indeed to determine which organism might be the most sensitive at detecting such a release).
  • the apparatus of the invention likewise provides for the simultaneous investigation of a broad spectrum of test agents on any given type of biological sample: a different test agent (e.g. solution or other medium) is contacted with each of a plurality of identical samples in the assay.
  • a different test agent e.g. solution or other medium
  • the spectrum of different test agents may even include the same material at a range of different concentrations, thereby permitting a concentration-response curve to be established.
  • the multi-well format it is even possible to monitor, in parallel, a plurality of different biological samples exposed to a plurality of different test agents.
  • One relevant use for the apparatus of the invention is in the high throughput screening of chemicals, e.g. to fill gaps in toxicity data (e.g. for the risk assessment of high production volume chemicals [HPVs]), e.g. in the development of drugs and pharmaceuticals.
  • toxicity data e.g. for the risk assessment of high production volume chemicals [HPVs]
  • HPVs high production volume chemicals
  • Historical data indicate that about 10,000-25,000 novel molecules are synthesised in order to produce a single compound which passes all the hurdles to reach the market place.
  • a significant number of compounds are withdrawn for toxicological and/or (in the case of agrochemicals especially) ecotoxicological reasons.
  • Carefully targeted short-term parallel screening tests can significantly improve the possibility of obtaining successful candidates, by winnowing out those with unacceptable structural and/or toxicological properties early on.
  • the apparatus of the invention may be used for range-finding, i.e. determining a suitable dilution range at which a particular test compound should be administered to a higher organism, again reducing the necessity for repeat testing in animals. It may also be used for prioritising a group of test agents for subsequent analysis, e.g. in vivo.
  • the apparatus of the invention is adapted for high throughput screening by comprising inter alia a plate having a plurality of wells.
  • the plate may comprise any convenient number of wells. It may have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more wells. It may have at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more wells.
  • the plate may have from 5-150, 5-125, 5-100, 10-150, 10-125, 10-100, 15-150, 15-125, 15-100, 20-150, 20-125, 20-100, 25-150, 25-125, 25-100, 30-150, 30-125 or 30-100 wells.
  • the wells may be arranged in a regular pattern. They may take any convenient size and shape. They may be rectangular (e.g. square) or circular in cross-section. They may define a substantially cylindrical or substantially hemispherical space.
  • the wells in the plate may be identical e.g. as to shape and/or volume, but that is not essential.
  • the apparatus may for example be adapted for simultaneously analysing a plurality of different test agents having different volumes. Two or more different shapes and/or sizes of wells may therefore be provided.
  • Any given well may have any convenient volume, as long as it is sufficient to hold a biological sample, e.g. at least one cell. It may have a volume of e.g. at least about 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 3000, 4000 or 5000 ⁇ l.
  • It may have a volume of from about 5 to about lOO ⁇ l, about 5 to about 200 ⁇ l, about 5 to about 300 ⁇ l, about 5 to about 400 ⁇ l, about 5 to about 500 ⁇ l, about 5 to about lOOO ⁇ l, about 5 to about 1500 ⁇ l or about 5 to about 2000 ⁇ l.
  • the apparatus may comprise 96 wells in an e.g. 8 12 array.
  • the apparatus may conform to the standard 96-well format conventionally used in the art, e.g. i the fields of microbiology and biochemistry.
  • the apparatus may be capable of interfacing with other devices employing the 96-well format. It may be capable of interfacing with e.g. an automatic pipetting device for simultaneously loading the 96 wells with one or more samples or reagents. It may be capable of interfacing with e.g. a machine for analysing the contents of each well by an assay technique other than one based on electrochemistry, as discussed elsewhere herein.
  • a plurality of wells is provided, each of which has a working electrode which is exposed to its interior.
  • the presence of other wells, which are not associated with an electrode, is not however excluded.
  • These 'non-electrode' wells may be used for analytical techniques other than chemically mediated amperometry.
  • optical analysis of the well contents may be used, e.g. by measurement of absorbance, luminescence or fluorescence.
  • a form of analysis other than chemically mediated amperometry may be used, either as an addition, or as an alternative to that technique.
  • the electrode will be configured, and the plate constructed, such that the alternative form of analysis is enabled. Electrode configuration and plate design are discussed elsewhere herein.
  • a further advantage of the present invention's capacity for simultaneously testing a large number of agents/samples in parallel is that each individual assay may be conducted under substantially the same conditions, e.g. as to temperature, pressure, humidity, oxygen and carbon dioxide levels, and the like.
  • each individual assay may be conducted under substantially the same conditions, e.g. as to temperature, pressure, humidity, oxygen and carbon dioxide levels, and the like.
  • the plate of the apparatus may comprise a substantially solid block, into which the plurality of wells is sunk.
  • the plate may comprise a support, e.g.
  • the support may provide a common base to the plurality of wells, the remaining wall(s) of each well being configured as a tube, e.g. of circular or rectangular cross-section, that is upstanding from the support.
  • the support may comprise a web including a plurality of holes. The holes provide the mouths of the wells, a wall or walls of each well depending from the support, from around a respective hole.
  • wall(s) may be mounted to, or integral with, the support. They may terminate in a closed base that is individual to each well, or may again take the form of a tube, with a connected or integral substrate providing a common base.
  • the support connects the wells together, whether or not it provides their base.
  • any suitable material may be used to form the plate, or one or more of its components, e.g. the walls of the wells and/or any support and/or any base substrate.
  • a ceramic material may be used.
  • the plate, or one or more of its components, e.g. abase of the wells maybe transparent. This will permit optical analysis of the well contents.
  • Suitable materials include acetate, polystyrene and polycarbonate.
  • the material of the plate, or at least that which provides the walls (including the base) of the wells, will preferably be chemically and/or biologically inert. In particular, it may be non- toxic to one or more of the types of biological sample described elsewhere herein.
  • Bonding substances may be used to secure one or more components of the apparatus together, e.g. the walls of the wells to a common base.
  • a bonding substance should preferably be chemically and/or biologically inert.
  • welding techniques e.g. heat sealing or radio frequency welding
  • Each well is associated with a working electrode for use in electrochemistry, e.g. for use in chemically mediated amperometry. At least a part of the electrode is exposed to the interior of the well.
  • the electrode may be entirely contained within the well, and the apparatus may comprise conduction means, e.g. a wire or printed conductive (e.g. metallic) track, for providing a path of electrical communication from the outside the well, to the electrode.
  • the electrode may extend into the interior of the well by passing through its mouth.
  • the electrode may extend through a wall of the well, e.g. through a base of the well, such that its proximal end is exposed to and/or protrudes into the well interior. The distal end of the electrode is exposed to and/or located outside of the well.
  • a substantially water-tight seal may be provided between the electrode/conduction means and the wall.
  • a seal may be formed by intimate contact between the electrode or conduction means and the wall.
  • a welded seal may be used.
  • a separate sealing means may be provided, e.g. a cured resin, a gasket etc.. Any sealing component exposed to the interior of the well may be chemically and/or biologically inert.
  • a plurality of working electrodes is provided on a support or substrate forming a common base to the plurality of wells. The remaining wall(s) of each well, which may be circular or rectangular in cross-section, will project from the common base.
  • a perforated web support may connect together, e.g. be integral with, the ends of the tubes which define well mouths, the interior of each tube being located in register with a respective hole in the web.
  • Electrodes may be provided on a common base by e.g. screen-printing, the base optionally being pre-scored.
  • the base may be made of ceramic, glass or a polymeric material. Any suitable ink or other material may be used for the printing, e.g. gold, copper, a silver/silver chloride ink or a high conductivity carbon-based ink.
  • the non-working areas of the base may be covered with an electrically insulating layer, e.g. a dielectric layer, which may be printed using a non-conducting polymer.
  • the electrodes may be electrically conductive pins or lugs which are mounted to and/or extend through the base.
  • the apparatus of the present invention may comprise one or more reference electrodes, against which the potential of one or more associated working electrodes can be measured.
  • Reference electrodes may be provided as combined reference/counter electrodes as is known in the art, but in certain embodiments of the present invention, one or more dedicated counter electrodes, separate from the reference electrode (s), may alternatively be provided (thereby forming a three electrode system).
  • working and reference electrodes are screen-printed onto a common base.
  • Electrode layouts may be designed on computer, e.g. using CAD or Gerber software.
  • Working and/or reference and/or counter electrodes may take any suitable configuration.
  • the electrodes will be structured appropriately.
  • an O- or C-shaped electrode may be appropriate. This will permit a central, unobstructed portion of the base, through which optical analysis may be conducted.
  • a reference electrode or other electrode
  • the working and reference electrodes may then be located concentrically, again providing a central, unobstructed portion of the base.
  • the working electrodes of different wells should ideally be maintained in electrical isolation, both from one another, and also from any reference electrode (s) .
  • the individual responses of biological samples contained within the different wells can then be monitored.
  • the apparatus comprises a plurality of reference electrodes, e.g. one associated with each well that has a working electrode, then these reference electrodes (or groups of them) maybe connected together. They may then be poised at the same potential. Electrical connection is achieved using conduction means. Unconnected electrodes may have different electrical potentials applied thereto.
  • the apparatus may comprise conduction means for providing a path of electrical communication from the outside the well to the electrode.
  • the apparatus may contain one or more electrical terminals for connecting the electrodes to one or more electrical devices for controlling, monitoring, and optionally recording, their potential.
  • the electrical terminals maybe mounted on the plate, e.g. at an edge thereof.
  • Conduction means may be provided for establishing a path of electrical communication from the electrodes to the terminals. Such paths may be provided with electrical switches.
  • any conduction means well known to those skilled in the art may be used.
  • a metallic or carbon-based conductor e.g. in the form of a wire, may be suitable.
  • a conductive track, screen-printed onto the plate, e.g. onto a support or base substrate, may be employed.
  • the use of copper conductors mounted on e.g. the support or base substrate (where present), may be advantageous. This is because of the length of electrical connections between the electrodes and/or terminals: carbon and/or silver-silver chloride inks, under certain circumstances, may not conduct low currents effectively over long distances.
  • Production of copper tracks may be performed using known methods of manufacture of printed circuit boards (PCBs).
  • PCBs printed circuit boards
  • a high speed lithographic printing technique may be used to provide conductive tracks on e.g. a support or base substrate.
  • the conductive tracks are printed directly onto the substrate/support in the desired configuration.
  • the process uses specialised inks or other materials, which can be used as conductors themselves, or as a base for subsequent plating to form the conductive tracks.
  • the technology may achieve finer line width and spacing than etch technology, and the circuits can be printed on any substrate, including a range of plastics.
  • Lithographic technology may increase the speed of production of the apparatus of the invention, whilst dramatically reducing the amount of copper or other metal required, and the overall cost of producing the apparatus. Furthermore, transparent materials may be used for the support/base substrate, thereby permitting optical analysis of the well contents. The risk of chemical contamination may also be reduced, as compared to etch technology.
  • conduction means are provided on a base component which is common to the wells, e.g. a support or additional base substrate, then those means may be carried, e.g. printed, on either side of the base.
  • An electrical connection between any two conduction means on opposite sides of the base may be achieved using e.g. drilled holes (through-holes or vias) which are plated.
  • the apparatus may comprise positioning means for determining (and optionally adjusting) the relative positions of (including e.g. the distance between) a biological sample and its respective working electrode within at least one (and preferably each) of the wells.
  • the apparatus in order to effect such control of position, may comprise, in addition to the plurality of wells, at least one insert for removably mounting a biological sample within a respective well.
  • the apparatus may in fact comprise, in addition to the plurality of wells, a corresponding plurality of inserts for removably mounting a biological sample (and optionally adjusting its position) within each of said wells.
  • control may be performed either uniformly across the wells, individually for each well, or otherwise non-uniformly, e.g. differently for each biological sample, differently for each test substance, or in a deliberately graduated fashion for a given biological sample or a given test substance, or otherwise. Greater flexibility may be achieved by means of, e.g. the permanent modification of preformed inserts, the use of alternative inserts having fixed dimensions, and/or the use of inserts having adjustable dimensions.
  • An insert may be constructed so as to permit the passage of a chemical mediator, e.g. a redox coupling reagent, between the biological sample and the working electrode. It is nevertheless capable of retaining the biological sample, thereby to permit its removal from the well at the end of the analysis.
  • a chemical mediator e.g. a redox coupling reagent
  • a portion or component of the insert may be porous, the holes being sufficiently large so as to permit the passage of a chemical mediator, but not the biological sample.
  • a porous portion or component of the insert may comprise or consist of a membrane.
  • the exact configuration of an insert may depend on e.g. the volume of the well for which it is intended, and the nature of the biological sample which it is designed to carry.
  • the insert may be configured for example as a cup, into which the biological sample, one or more test agents and a chemical mediator may be placed in use.
  • the cup may be adapted so it can be mounted in the well such that its lower most portion is inside the well, but spaced a predetermined distance from the well base. This may be achieved using a shoulder or lip of the cup which is capable of resting on a portion of the plate which defines the mouth of the well. This portion of the plate may be e.g. an end of a wall of the well or a section of the support.
  • the cup may comprise one or more legs which in use contact the base of the well.
  • a shoulder, lip, leg or other means of predetermining distance from the well base may be adapted to enable adjustment of that distance, for example by permanent modification, interposition of an additional component, or a built-in telescopic or ratchet mechanism
  • an insert is preferably chosen so as not to obstruct the operation of any electrode (s) in the well.
  • any electrode s
  • the cup is provided with legs, then these should be arranged and/or configured so as not to contact the electrode.
  • the inserts may be suitable for complete immersion within the contents of a well, whilst still preventing an escape of the biological sample.
  • a membranous bag may be used.
  • the inserts may be beads.
  • the inserts may be made of any appropriate material, but that material is preferably electrically non-conductive and chemically and/or biologically inert. The materials described elsewhere herein for manufacture of the plate may be used. Where the apparatus is designed for optical analysis of the well contents, the inserts maybe made of a transparent material, e.g. of acetate, polystyrene or polycarbonate.
  • inserts greatly enhances the versatility of the apparatus of the present invention.
  • a biological material e.g. cells, or viruses on a cell lawn
  • Steps involved in the transfer of cells from an established culture to the sensory apparatus can therefore be avoided. Even where the culture is a liquid, such transfer may be detrimental to the cells. It may involve steps such as centrifugation, which may damage and/or alter the behaviour of the cells.
  • inserts may facilitate the use of eukaryotic cells, especially animal cells, as the biological sample.
  • Animal cells are typically grown as a monolayer anchored to a culture dish; the use of an insert (or component thereof) on which the cells are grown, will obviate the need for removing the cells from that dish. Since such removal may comprise harmful and/or behaviour-altering steps, e.g. treating the cells with enzymes (e.g. trypsinisation), the integrity of the cells will be substantially preserved. Exposure of the cells to enzymes may also be time consuming.
  • enzymes e.g. trypsinisation
  • an insert further permits a biological sample to be returned to culture, after its , analysis, and optionally re-examined. This allows recovery studies to be conducted. It also provides for an investigation as to whether a given test agent (or concentration thereof) causes sensitisation or desensitisation of the biological sample: repeat exposure and monitoring of the same biological sample may be conducted periodically.
  • the inserts of the plurality may be discrete from one another such that any given insert may be removed from its respective well, without disturbing the others. Alternatively, at least some of the inserts may be mechanically coupled or fused together, thereby forming a set of inserts to facilitate their co-ordinated manipulation. In embodiments of the invention in which the plurality of wells is arranged in an array, inserts may be joined or fused together to reflect the structure of a regular part thereof, e.g. a row or column.
  • the apparatus of the invention may be provided with contacts or terminals for connecting the electrodes to electrical devices. Such devices may be used for e.g. monitoring and optionally recording the potentials of the working electrodes. They may be used for e.g. maintaining the potential of the reference electrode (s) .
  • the apparatus may comprise a multi-channelled potentiostat for controlling the potential difference applied to the electrodes and collating the signals from each individual electrode on a computer.
  • a potentiostat will have a number of channels which ideally corresponds to (or exceeds) the total number of electrodes (or groups of electrodes) in electrical isolation from one another.
  • amperometry is to be conducted in each well of a 96-well plate (using the standard 96-well format known in the art) , then a potentiostat having at least 96 channels for 96 working electrodes, and at least one channel for each reference or counter electrode (or group of such electrodes) is required. Each channel is in electrical communication with one of the electrodes. Potentiostats may be obtained from e.g. Whistonbrook Technologies Limited.
  • the apparatus of the invention may comprise data processing means, e.g. a computer and appropriate software, for collecting, storing and optionally recording the potential of each working electrode relative to an associated reference electrode.
  • the computer may further permit data display and/or analysis. Connection between a computer and a potentiostat may employ any conventional means.
  • the apparatus of the invention may be sufficiently small so as to be easily transportable between laboratories and/or useable in the field, e.g. where on-site analysis of environmental or clinical samples is required.
  • a mobile power source e.g. a battery pack, may be provided.
  • a potentiostat may be incorporated into, e.g. mounted on, the plate.
  • One or more components of the apparatus of the invention e.g. the plate and/or the inserts (where present) which may comprise adjustment means for adjusting the location of a biological sample relative to a respective working electrode, may be disposable.
  • the component (s) may be capable of being sterilised by one or more methods, without impairment of function.
  • the apparatus of the invention or at least the plate component and/or inserts thereof, is capable of being sterilised.
  • the apparatus of the invention may be capable of being sterilised by any of the above .methods, or a combination thereof, without impairment of function.
  • the desire for a sterilisable apparatus should be taken into account when selecting the materials for its manufacture, including any screen-printing inks or adhesive. Such selection is well within the knowledge of the person of skill in the art.
  • the apparatus may comprise a lid or other covering means for overlying the mouth of at least one well, (preferably all wells) thereby to prevent (or at least inhibit) the entry of micro-organisms.
  • the covering means may be provided with securing means for mounting it on the plate. Any conventional securing means may be employed, whether mechanical, e.g. a friction fitting, or chemical, e.g. an adhesive.
  • the present invention provides a method of monitoring how a plurality of biological samples responds to one or more test agents, the method comprising the steps of contacting each sample with an electron transfer mediator and a test agent in the presence of an electrode, and monitoring in parallel the transfer of electrons from the samples to their respective electrodes by the mediators.
  • the transfer may be monitored by measuring the electrical potential of each electrode relative to a respective (or common) reference electrode.
  • the positions of the biological samples relative to their respective working electrodes maybe controlled. This may be achieved using positioning means as described elsewhere herein. Different biological samples may be positioned at different distances from their respective working electrodes.
  • the method of the invention may be based on chemically mediated amperometry, as discussed elsewhere herein.
  • Examples of appropriate chemical mediators and electrolytes are well known to those skilled in the art. Reference is made to e.g. Evans, M. R. et ah Pesticide Science 54, pp.447-452 [1988] .
  • the chemical mediators p-benzoquinone and 2,6-dimethyl-benzoquinone may be mentioned.
  • the method may further comprise a step of contacting an identical biological sample with an identical electron transfer mediator in the absence of the test agent.
  • the transfer of electrons from the sample to an identical electrode is monitored, and the transfer is compared to the transfer recorded in the presence of the test agent.
  • Monitoring of identical samples in the presence and absence of test agent may be conducted simultaneously, i.e. in parallel, e.g. in adjacent chambers of the same apparatus. This may help to eliminate variables such as temperature, oxygen and carbon dioxide levels.
  • the transfer of electrons observed in the presence of test agent may be compared to pre-recorded data showing the transfer of electrons from the sample in the absence of test agent.
  • the effect of a given test agent on a particular biological sample may be determined by: (i) contacting the sample with an electron transfer mediator in the presence of an electrode; (ii) monitoring the transfer of electrons from the sample to the electrode by said mediator, thereby to establish a basal activity for the sample; (iii) contacting the sample with a test agent; and (iv) monitoring the transfer of electrons from the sample to the electrode by the mediator in the presence of the test agent, thereby to determine a test activity for the sample. The basal and test activities of the sample may then be compared.
  • the method of the invention may comprise such steps for two or more of the plurality of biological samples being monitored. It may comprise those steps for all of said samples.
  • a basal activity need not always be determined.
  • a plurality of identical biological samples may be exposed to a respective plurality of test agents comprising the same substance at a range of different concentrations.
  • the zero concentration activity may be determined by extrapolation.
  • the electrochemical component of the assay is designed for continuous monitoring.
  • the rate of action of a given test compound e.g. its rate of toxic action, can therefore also be determined.
  • the method of the invention may thus comprise a time period during which the transfer of electrons from the biological samples to their respective electrodes is continually, or periodically, monitored.
  • the test agents are contacted with the samples at a particular point in the test period, and the change in electron transfer (if any) which results from the test agents is recorded.
  • the rate of change of the measured 'current' is determined and optionally analysed.
  • the method may be used to determine the recovery of a particular biological sample, after its exposure to one or more test agents.
  • the method may comprise the steps of: (i) contacting at least one of said biological samples with an electron transfer mediator and a test agent in the presence of an electrode; (ii) monitoring the transfer of electrons from the sample to the electrode by the mediator, thereby to determine a test activity for the sample; (iii) removing the test agent from the sample; and (iv) periodically or continuously monitoring the transfer of electrons from the sample to the electrode by the mediator, after the test agent has been removed. Removal of test agent may occur by any conventional technique known to those skilled in the art, e.g. by aspiration, filtration, etc.
  • the method may comprise the steps of contacting the sample with the electron mediator in the presence of the electrode, and monitoring the transfer of electrons from the sample to the electrode by said mediator, thereby to establish a basal activity for the sample.
  • the extent of recovery of the sample may be more easily determined, e.g. by comparison of the transfer of electrons monitored in step (iv), with the previously measured basal activity.
  • the sample may be removed from the apparatus in which the electrode is located, and returned to conditions for cell culture, e.g. to an incubator.
  • the apparatus may comprise removable inserts or carriers on which, or within which, the sample is retained. In that way, the sample does not need to be subjected to treatments which might cause damage and/or a disturbance in behaviour, e.g. treatments for releasing anchored cells from a culture plate.
  • removable inserts in association with electrochemistry, e.g. in association with chemically mediated amperometry, is discussed elsewhere herein, within the context of the present invention, more particularly the apparatus of first aspect of the invention.
  • Removable inserts may also facilitate methods of the invention in which an investigation is conducted as to whether a given test agent (or concentration thereof) causes sensitisation or desensitisation of a biological sample: repeat exposure and monitoring of the biological sample may be conducted periodically, with the sample being returned to culture conditions in between times.
  • the method involves monitoring a plurality of biological samples in parallel by electrochemistry, e.g. by chemically mediated amperometry.
  • a sensory apparatus comprising a plurality of chambers, each of which is associated with a working electrode may be used. If a respective plurality of removable, discrete inserts is associated with the plurality of chambers, then it becomes easier to manipulate any particular assay without disturbing the others.
  • a first set of chambers in the sensory apparatus may be used to monitor how a first plurality of different biological samples reacts to a particular test agent.
  • a second set of chambers in the same apparatus may be used to monitor how the same spectrum of samples reacts to a different test agent (e.g.
  • a third set of chambers may be used to monitor recovery of a third plurality of biological samples, following their exposure to a further test agent. Whilst electron transfer is continually monitored in the first and second set of chambers, the third plurality of samples may be loaded into the third set of chambers on inserts, their electron transfer to respective working electrodes monitored, and the samples returned to culture.
  • the apparatus employed in the method of the invention may comprise a series of electrical switches for reversibly completing paths of electrical communication between electrodes and terminals.
  • Any method of the present invention may involve the use of any apparatus according to the first aspect of the invention. Various features of such apparatus are described elsewhere herein, and will not be repeated here.
  • an apparatus employing the established 96-well format may particularly be mentioned.
  • Such an apparatus may be based on a modified microtitre plate. In that way, only small sample volumes are required. This may be important where the quantity of the test agent(s) available for analysis is limited (e.g. in drug development).
  • a sensory apparatus which comprises a plate having a plurality of sampling areas, each of which is associated with a working electrode.
  • a biological sample and a test agent are located onto each sampling area, over the electrode.
  • the sample and agent may be spotted onto the sampling area and held in place by surface tension.
  • Such an apparatus is particularly suitable for use with small sample volumes of sample, e.g. in the field of proteomics.
  • the apparatus may include any of the features (or combinations of features) that may be found in the apparatus according to the first aspect of the invention, unless the context requires otherwise.
  • control of the relative positions of a sample and its respective working electrode may be achieved by floating a suitable membrane or other surface carrying the sample, on a spot of fluid retained over the electrode by surface tension.
  • the fluid may contain the test agent and/or mediator.
  • a membrane or surface maybe distributed across the surface of the plate either as a continuous layer (itself being so composed and fabricated as to prevent mixing between the fluid volumes in contact with the working electrodes) or as discrete units, one for each location of a working electrode. Fluid or fluids containing, e.g. the test agent and/or the mediator may then be spotted onto the membrane or surface.
  • the positioning of the membrane or surface relative to its respective working electrode (s) may be respectively adjusted by varying the volume of the retained spot of fluid, or by shaping the membrane or surface so as to determine its clearance in relation to each electrode that it covers.
  • the membrane or surface may contain the biological sample, thereby preventing any direct physical contact between the sample and the electrode, or the biological sample may adhere to one or both sides of the membrane or surface.
  • the method of the invention may involve, in addition to analysis by electrochemistry, e.g. chemically mediated amperometry, which may be conducted using the apparatus of the first aspect of the invention, one or more alternative analytical techniques, e.g. assays based on optical, e.g. UV, visible, fluorescent or luminescent endpoints, e.g. where a compound undergoes a colour change due to its reduction oxidation by the sample (or by a mediator previously reduced/oxidised by the sample) .
  • the assays may be conducted at the same time as the amperometry, either on biological samples which are simultaneously being monitored by amperometry, or on parallel samples, e.g. samples contained within separate wells of the same apparatus, or on both types of sample. Through the use of multiple parallel analytical techniques, an increased amount of information can be collected in the same test run. The throughput efficiency of the analytical system is therefore improved.
  • Examples of alternative assays include the fluorescein leakage test (which employs cells with tight junctions in order to detect subtle junctional defects) , the resazurin test (a cell viability test), and the Neutral Red and Alamar Blue assays.
  • One alternative analytical technique employs biological cells which have been engineered to contain reporter genes. A reporter gene may provide for an additional and unrelated activity of a biological sample, which may be modified in response to a test agent.
  • Reporter genes may code for biological molecules possessing unique properties which are easily distinguishable from endogenous cellular functions. They can generate, e.g. colorimetric, fluorescent, luminescent, • chemiluminescent or electrochemical signals, which may be proportional to the concentration of the test agent to which their transgenic hosts are exposed.
  • luminescence may be achieved using the lux genes encoding luciferase enzymes. Such enzymes catalyse light-emitting reactions, and are commonly found in a range of bioluminescent (light-emitting) organisms. There are five lux genes responsible for the light-emitting reaction. If all five genes of the lux cassette are incorporated into a test cell, then a completely independent light producing system is created, requiring no additional substrate to be added, and no excitation by an external light source. Luminescent reporter genes may be used to determine the metabolic status of the cells, following chemical challenge. Disturbances in luminescence illustrate e.g. toxic effects of test agents which may result from e.g. transcriptional inhibition.
  • Reporter genes can provide a robust, cost-effective, quantitative method for the rapid and selective detection and monitoring of chemical and biological test agents. Similar to . chemically mediated amperometry, reporter technology can often be implemented in realtime, on-line bioassays, with intact, living cell systems, thereby providing a unique and revolutionary perspective on bacterial, plant and mammalian physiology, including cellular interactions.
  • the technology may be used in the drug discovery process to study e.g. transcriptional regulation in response to a test agent. It may be used for secondary pharmacological and receptor binding assays.
  • the methods and apparatus of the present invention may be used in a diverse array of detection processes and behavioural studies, e.g. in methods of medical diagnostics, precision agriculture, environmental monitoring, food safety, process monitoring and control, and potential pharmaceutical analysis. Such uses in fact provide yet further aspects of the present invention. Particular examples of such uses are described elsewhere herein.
  • the following provides yet further illustration, by specifying examples of the biological samples and/or test agents that may be employed. In general, the invention is useful where it is desired to establish whether (and optionally to what extent) a given test agent affects a particular biological sample.
  • the plurality of biological samples maybe identical to one another, e.g. in the event that a user wishes to determine how a plurality of different test agents affects one particular cell type or tissue.
  • the spectrum of test agents may even include the same material at a range of different concentrations, thereby permitting a concentration-response curve to be established.
  • test agent may be a pure or substantially pure compound, e.g. in embodiments where the high throughput screening of individual chemicals is to be conducted.
  • Such methods may be used in the risk assessment of high production volume chemicals (HPVs) , or in the development of drugs and pharmaceuticals, as discussed elsewhere herein.
  • HPVs high production volume chemicals
  • the method may be used for the rapid testing of a large number of potentially therapeutic compounds generated by combinatorial chemistry.
  • the test agent may comprise a mixture of substances.
  • the test agent may include a material taken from the environment, e.g. when the method of the invention is used to detect pollution and/or to monitor dispersion and/or degradation of one or more pollutants.
  • An environmental material may be taken from e.g. soil, rock, sand, water (e.g. from a river, lake, ocean or underground water source). It may be taken from biological matter, e.g. it may comprise an extract or homogenate from a plant or animal, e.g. a specimen of blood, plasma, lymph, bile, gastric fluid, tissue fluid, urine, faeces, or the like.
  • Biomaterials may also used as test agents when the method of the invention is used in clinical analysis, e.g. where one or more isolated specimens (from the same or different patients) is used for a method of in vitro diagnosis. Any of the above-mentioned biomaterials may be used. Such materials, e.g. blood, may be pre-treated, prior to their use in the method of the invention, e.g. by the removal of cellular components, e.g. by centrifugation and/or filtration.
  • a test agent may be a foodstuff, e.g. an aliquot of allegedly purified drinking water.
  • a test agent may include a material obtained by or derived from an industrial process. It may include an industrial waste material (e.g. waste water) which may be destined for release into the environment.
  • test agent may be solid or liquid in nature.
  • Liquid test agents may be aqueous or non-aqueous, i.e. organic.
  • a liquid agent may be a solution, suspension, or emulsion.
  • a monitoring apparatus e.g. one affording control of the positioning of one or more (e.g. all) of the biological samples relative to their respective working electrodes, and in particular such an apparatus comprising a plurality of chambers, e.g. wells, each containing an electrode, and a respective plurality of removable carriers, e.g. inserts, may facilitate the use of test agents which are either: (i) solids of poor solubility; or (ii) non- aqueous liquids.
  • water-insoluble chemicals may be dissolved in non- aqueous solution (e.g. mineral oil) and placed in an insert which comprises a membrane and is loaded with a biological sample.
  • the chemical mediator and an electrolyte are placed in a respective chamber of the apparatus, and the insert is located within the chamber.
  • the membrane of the insert separates the two liquid phases but permits the passage of the chemical mediator.
  • confluent e.g. animal cells are grown, on the membrane, the cell layer will also participate in separating the two liquid phases.
  • a single test agent may be contacted with a plurality of different biological samples.
  • this may be appropriate where a given test chemical is suspected of being toxic to a particular animal, e.g. to a human, but the organ which is most affected is unknown: a range of different cells from the animal may be tested simultaneously.
  • an environmentally relevant chemical is to be tested against a range of different micro-organisms, in order to determine which (if any) species is at most risk, should a release of the chemical into the' environment occur.
  • cell-cell interaction may modify a toxic (or pharmacological) response, e.g. where a given cell type, which is relatively insensitive to the direct effect of a particular test agent, is more severely compromised by the lack of physiological signals received from a second cell type, which is directly affected.
  • a toxic (or pharmacological) response e.g. where a given cell type, which is relatively insensitive to the direct effect of a particular test agent, is more severely compromised by the lack of physiological signals received from a second cell type, which is directly affected.
  • the one or more biological samples may be selected from the group consisting of eukaryotic cells, prokaryotic cells, cellular organelles, membranes and mixtures thereof.
  • Eukaryotic cells include plant cells, animal cells and fungal cells. Algae or higher plant cells may be used.
  • Animal cells may be taken from mammals, e.g. from humans or other primates (e.g. from chimpanzees). They may be taken from agricultural animals such as horses, cows, goats, sheep, pigs, or birds (e.g. from chickens or geese) , or they may be taken from domestic animals such as dogs, cats, rabbits, and the like.
  • Prokaryotic cells may be taken from any species of bacteria, e.g.
  • organelles and membranes may include those which comprise, or are associated with, one or more components of an electron transport chain, e.g. chloroplasts and mitochondria and membranes therefrom.
  • isolated proteins or mixtures of proteins may be used as a biological sample, e.g. cytochromes, photosynthetic pigments, and the like.
  • Biological samples may comprise transgenic cells, i.e. cells containing a heterologous nucleic acid sequence such as a reporter gene.
  • the transgene may provide for an additional and unrelated activity of the biological sample which may be modified in response to the presence of a test agent.
  • Biological samples may take any suitable form known to those skilled in the art, e.g. cell suspensions or cell layers grown on removable carriers or inserts of the sensory apparatus, or on components thereof, e.g. on membranes. Multiple cell layers and back-to-back cultures may be used, as discussed elsewhere herein.
  • cytotoxicity Notwithstanding the concept of basal cytotoxicity, it may be desirable to examine the effect of a particular test agent on an in vitro biological system comprising more than one cell type. Although the investigation of cytotoxicity is an important aspect of the present invention, the methods and apparatus described herein are not so limited. Moreover, (and as explained previously) , cell-cell interaction may influence the toxic effect of a compound in vivo, although it may ultimately act on a basic cellular metabolic function. researchers have noted that many cells rely on the presence of other cell types in order to function correctly.
  • a range of cell-cell interaction factors e.g. cytokines and growth factors
  • cytokines and growth factors are known to regulate a wide array of cellular functions, including cellular defence and repair systems, which can affect the toxic or pharmacological response in an organism, subsequent to its exposure to one or more chemicals.
  • Cell culture inserts may be used to grow back-to-back cultures. Different cell types may be grown on each side of a micro-porous insert membrane, enabling cell-cell contacts or contact through soluble factors (Van Gompel, J : Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. Balls, M. et al) pp. 231-236. Elsevier, Oxford [2000]).
  • Using chemically mediated amperometry optionally in conjunction with optical endpoints, it is possible to monitor the effects of intercellular interactions, in particular by comparison with the results of assays on individual cell types.
  • liver toxicity and the identification of metabolic pathways in the liver, are important aspects in the characterisation of the toxicity of chemicals in humans.
  • Kupffer cells in the liver are known to release a number of intercellularly-acting mediators which are suspected of modulating the effects of hepatotoxic compounds, by intercellular signalling directed towards hepatocyte cells (Maier, P: Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation, supra).
  • the identification of potentially neurotoxic activity may also be an important aspect of toxicity assessment.
  • the neurons in the brain which receive, integrate and transmit information only account for 10% of brain cells. Glia (the majority of brain cells) provide mechanical support, regulation of the extracellular environment, production of cytokines and neurotrophins, and regeneration, and are thought to exacerbate neurotoxicity. Co- cultures could be used to bring cell types such as cortical neurons, astrocytes and microglia together. The results may be compared with pure cultures to determine cell relationships, e.g. to determine whether there is an increase or reduction in toxicity when the cells are cultured together (Kirkpatrick, C.
  • liver slices have been used in recent investigations of drug metabolism and liver toxicity because all the cells are present in their natural state, with their original cell-cell contacts intact. It is therefore believed that they may be more discriminating for compounds possessing intricate mechanisms of toxicity. They may be useful for the examination of chronic or long term toxicity.
  • hepatocytes in a liver slice are closer to hepatocytes in the liver than are isolated hepatocytes.
  • Precision-cut liver slices are now commercially available. These are highly reproducible and stable (lasting up to 5 days). 75 slices may be obtained from a single rat liver and 20,000 from a human liver. These slices are each approximately 8- 10mm diameter, which is sufficient volume for a detailed study (Bach, P. H.
  • tissue slices for pharmacotoxicology studies. The report and recommendations of ECVAM workshop 20. ATLA pp. 893-923 [1996]).
  • a plurality of tissue slices may be placed in a plurality of culture inserts, or otherwise positioned relative to respective electrodes for monitoring a plurality of biological samples, and immersed in culture media or perfusion solution, in the presence of a mediator for amperometric monitoring.
  • Figure 1 shows examples of electrode arrays screen printed onto a ceramic base.
  • Figure 1(a) shows an 8 well linear arrangement as used in Experiment 1.
  • the working area is shown in more detail in Figure 1 (b) , where the black area is the working electrode and the grey area is a silver/silver chloride reference electrode.
  • Figure 1 (c) shows the design as applied to the 96-well format.
  • Figure 2 shows a comparison of the amperometric signal resulting from bacteria placed directly in a well, with the signal produced by bacteria contained in an insert. Bacteria were present in the insert at time 0, whereas bacteria were added to the well at time 180s. A control containing no bacteria is also shown.
  • Figure 3 shows a comparison of the amperometric signal resulting from bacteria placed directly in a well, with the signal produced by bacteria contained in an insert. Bacteria were present in the insert at time 0 whereas bacteria were added to the well at time 180s. A high concentration of the toxicant ZnS0 4 was added to the insert at time 1840s, resulting in a reduction of the bacterial signal to baseline.
  • Figure 4 shows the amperometric signals obtained from bacteria exposed to different concentrations of the toxicant 3,5-dichloro ⁇ henol.
  • Figure 5 shows a plot of percentage inhibition of bacteria vs. concentration of 3,5- dichlorophenol.
  • the apparatus and methods of the present invention maybe used to monitor cultured mammalian cells in a plurality of wells of a 96-well microtitre plate, for high throughput screening applications.
  • the present experiment demonstrates that the use of culture inserts has no problematic effect on the technique of chemically mediated amperometry, e.g. because the chemical mediator may be required to pass through a porous component of the insert, e.g. a membrane, in order to interact with the cells and thereafter pass back to the working electrode.
  • the response from bacteria held fn a culture insert was used as indicator of the approximate amperometric signal amplitude and profile which might be expected from mammalian cells cultured on the membrane of the insert.
  • the maximum speed of response and signal amplitude was also determined for a suspension of bacteria placed directly into a well, without the use of an culture insert (this would not be possible using an anchorage-dependent mammalian cell line).
  • the parameters with and without culture inserts were compared, in order to determine to what extent the flow dynamics and mixing within the wells are affected by the inserts.
  • a plate shaker was used to promote mediator transfer.
  • Nutrient broth for bacterial culture was prepared by dissolving 5 g of nutrient broth no. 2 (Oxoid Ltd., Basingstoke, UK) in 200 ml of ultra-pure water. This was divided into 4 ml aliquots in McCartney bottles (The Microbiological Supply Company, Toddington, UK) and autoclaved at 121 °C for 15 minutes in order to ensure sterilisation.
  • a glycerol suspension of a non-pathogenic K12 strain of Esc ericlr a coli bacteria was obtained from the Sensor and Cryobiology Unit at Luton University.
  • Physiological saline solution (0.85% w/v) was prepared by dissolving one saline tablet (Oxoid Ltd., Basingstoke, UK) in 500 ml of ultra-pure water..
  • a vial of freeze-dried bacterial substrate solution (Cellsense Ltd., Cambridge, UK) was reconstituted by injecting 10 ml of the saline into the vial and agitating until all substrate was dissolved.
  • Working strength saline substrate medium (SSm) was prepared by making the solution up to 500 ml with saline.
  • the sub-cultured bacterial suspension was adjusted to obtain an optical density of 1.6 at 430 ran using a spectrophotometer which had been zeroed using nutrient broth as a blank.
  • a 1 ml aliquot of the suspension was then centrifuged at 6000 rpm for 4 min to obtain a bacterial pellet.
  • the supernatant was removed and the pellet reconstituted in 1 ml of saline. Centrifugation and resuspension was performed again twice, with the final reconstitution being in 250 ⁇ l of SSm/mediator solution.
  • a stock mediator solution had been prepared on the same afternoon by dissolving 5.4 mg of p r ⁇ -benzoquinone (pBQ) (Sigma-Aldrich Company Ltd., Poole, UK) in 10 ml of SSm.
  • the SSm/mediator solution was then prepared immediately before the final bacterial resuspension by adding 100 ⁇ l of the f ⁇ r ⁇ -benzoquinone to 750 ⁇ l of SSm.
  • Amperometric monitoring of the E. coli bacterial cells was conducted in an electrode/microplate construct (secured on a microplate shaker at 600 rpm) coupled to an Autolab PGSTAT10 single channel electrochemical workstation (Windsor Scientific Ltd., Slough, UK).
  • 15 ⁇ l of bacterial solution was placed in an insert having a 0.2 ⁇ m anapore insert membrane, and blotted to remove the solution, leaving a layer of bacteria on the insert membrane.
  • 15 ⁇ l of SSm/mediator was added to the insert (without disrupting the bacterial layer) in order to protect the cells from dehydration. It is also important that the insert membrane is saturated in order to prevent the production of air bubbles below the insert.
  • the insert was lowered into a well which contained 50 ⁇ l of SSm/mediator solution and amperometric monitoring initiated. Where no insert was used (i.e. a free solution of bacteria was tested), 15 ⁇ l of the bacterial solution was added to 50 ⁇ l of SSm/mediator solution in the well after 180 seconds of amperometric monitoring. A control was also run using 65 ⁇ l of SSm/mediator solution (no bacteria). The results are shown in Figure 2. This experiment was repeated in order to determine the ability of the system to detect the effect of a high concentration of a known toxic chemical on the signal, directly reflecting the effect of the toxicant on the bacterial cells. The results are shown in Figure 3.
  • the second experiment demonstrates that the effects of a high concentration of the toxicant zinc sulphate on the assay can be clearly observed, even at these low currents.
  • Extrapolation of the baseline (the signal produced when no bacteria were present in the well) provides almost an exact correlation with the bacterial response after several minutes of exposure to the toxicant. This suggests that the cells are no longer able to reduce the mediator, either because their electrochemical activity has been impaired or because they have ben killed by the exposure.
  • Example 1 demonstrates that a mediated amperometric signal maybe obtained from living bacterial cells contained in inserts suspended in electrode containing wells of a modified microtitre plate, and that the strength of the signal is dependent on the number of viable cells that are present.
  • the present experiment demonstrates that the strength of the signal derived from a fixed concentration of bacteria is reduced in response to an increasing concentration of a toxic chemical.
  • the experiment demonstrates that the amperometric assay may be used to compare the toxicities of different test agents. It also shows that the assay may also be utilised to determine the toxicity of an environmental sample compared to a corresponding unpolluted control sample.
  • the bacteria were again contained within culture inserts as this gives an indication of the speed of mediator transfer between cells and electrodes and thus gives an indication of the toxic response time for both bacterial cells and anchorage- dependent cells (e.g. mammalian cells) which may be grown on the insert, or a component thereof, e.g on a membrane.
  • anchorage- dependent cells e.g. mammalian cells
  • 96-well microtitre plates with no well bases were provided by Life Technologies Ltd (Paisley, UK) . These plates were carefully machined into 2 x 8 -well sections to which two screen-printed ceramic electrode strips were fixed using either a cyanoacrylate "superglue” (Loctite) or an epoxy-based (Araldite Rapid) adhesive. The constructs were left overnight to ensure total setting of the adhesives.
  • Nutrient broth for bacterial culture was prepared by dissolving 5 g of nutrient broth no. 2 (Oxoid Ltd., Basingstoke, UK) in 200 ml of ultra-pure water. This was divided into 20 ml aliquots in 100 ml conical flasks and autoclaved at 121 9 C for 15 minutes in order to ensure sterilisation.
  • a glycerol suspension of a non-pathogenic K12 strain of Escherichi ⁇ coli bacteria was obtained from the Sensor and Cryobiology Unit at Luton University. 50 ⁇ l of the glycerol stock was added to a 20 ml aliquot of nutrient broth and placed in a shaking incubator (200 rpm) at 37 9 C overnight.
  • resulting bacterial suspension was then sub- cultured by adding 500 ⁇ l of this overnight culture to another 20 ml aliquot of nutrient broth, and returning the resultant culture to the 37°C shaking incubator for 4 hours. This was performed in order to obtain a log phase bacterial population which would be expected to have the highest metabolic activity for testing.
  • Physiological saline solution (0.85% w/v) was prepared by dissolving a saline tablet (Oxoid Ltd., Basingstoke, UK) in 500 ml of ultra-pure water.
  • a vial of freeze-dried bacterial substrate solution (Cellsense Ltd., Cambridge, UK) was reconstituted by injecting 10 ml of the saline into the vial and agitating until all substrate was dissolved.
  • Working strength saline substrate medium (SSm) was prepared by making the solution up to 500 ml with saline.
  • the sub-cultured bacterial suspension was adjusted to obtain an optical density of 1.6 at 430 n using a spectroph ⁇ tometer which had been zeroed using nutrient broth as a blank. 10 x 1 ml aliquots of the suspension were then centrifuged at 6000 rpm for 4 min to obtain bacterial pellets. The supematants were removed and the pellets each reconstituted in 1 ml of saline. Centrifugation and resuspension were performed again twice, with the final reconstitution of all pellets being in a total of 500 ⁇ l SSm.
  • a stock mediator solution of 50 mM potassium ferricyanide (Sigma- Aldrich Company Ltd., Poole, UK) in SSm was prepared on the same day.
  • a 5 mM working mediator solution was then prepared immediately before preparing the bacterial resuspension by preparing a xlO dilution of the stock mediator solution in SSm.
  • a 1 g/L stock solution of the toxicant 3,5-DCP (3,5-dichlorophenol, Sigma- Aldrich Company Ltd., Poole, UK) was prepared in working mediator solution on the day of testing. This was used to prepare 25, 50 and 100 mg/L solutions of the toxicant in working mediator solution. A 0 mg/L control (working mediator solution only) was also set aside for testing.
  • Amperometric monitoring of E. coli bacterial cells was conducted in an electrode/microplate construct (secured on a microplate shaker at 500 rpm) coupled to a 16 channel potentiostat (Whistonbrook Technologies Ltd, Luton, UK) .
  • the potentiostat was connected to a PC loaded with appropriate software for use with the potentiostat.
  • Each of the 16 wells of the electrode/microplate construct was loaded with 50 ⁇ l of 0, 25, 50 or 100 mg L of 3,5-DCP in working mediator solution (four wells of each concentration) .
  • the inserts were then lowered into the wells so that the insert membranes were saturated. This ensured that no air was contained in the membranes which otherwise might have prevented mediator transfer.
  • 30 ⁇ l of bacterial solution was placed in two of the inserts within each group of wells of a particular toxicant concentration.30 ⁇ l of SSm only (no bacteria) was placed in the two remaining inserts of each group.
  • the microplate stirrer was turned on (500 rpm) and amperometric monitoring initiated. The assay was run for approximately 90 minutes.
  • Inhibition (%) ((1-corrected slope) /corrected slope) x 100

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Abstract

La présente invention concerne un appareil et une technique qui permettent de surveiller comment une pluralité d'échantillons biologiques répondent à un ou plusieurs agents de test. Cet appareil comprend une plaque de microtitration possédant une pluralité de puits, chacun d'eux étant associé à une électrode de travail qui est exposée à l'intérieur du puits. Pour un ou plusieurs de ces puits (tous, par exemples), les positions relatives d'un échantillon biologique dans le puits et son électrode de travail respective peuvent être commandées. Cette invention permet d'optimiser des procédures de dosage en fonction de la nature du ou des échantillons biologiques, du ou des agents de test et de l'objectif de ces dosages.
PCT/GB2003/001453 2002-04-04 2003-04-03 Appareil et techniques d'analyse biochimique WO2003087293A1 (fr)

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WO2015078884A1 (fr) * 2013-11-26 2015-06-04 Alleati Ag Procédé et ensemble microfluidique pour test de sensibilité aux antibiotiques
JPWO2017154801A1 (ja) * 2016-03-11 2019-01-10 パナソニックＩｐマネジメント株式会社 電気化学測定システム、電気化学測定装置および電気化学測定方法
WO2023217839A1 (fr) 2022-05-11 2023-11-16 Institut National Polytechnique De Toulouse Procede d'analyse de la viabilite de cellules animales par mesure electrochimique
US11846626B2 (en) 2017-11-09 2023-12-19 Battelle Savannah River Alliance, Llc Electrochemical detection of microbial stress

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